Determination of lipoprotein lipase activity in biological samples by high performance liquid chromatography with fluorescence detection

• Main Authors: Yu-Ching Chou, 周郁晴
• Other Authors: Jen-Ai Lee
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/01904583927842463777

碩士 === 臺北醫學大學 === 藥學系 === 93 === Lipoprotein lipase (LPL) is associated with the luminal side of capillaries and arteries where it hydrolyzes triglycerides in circulating lipoproteins to produce free fatty acids. It was valuable to determine LPL activity which was shown to vary in diseases and metabolic disorders. This research aim to develop a highly sensitive HPLC method using the fluorescent reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ), for derivatization of oleic acid (OA) liberated after triolein being hydrolyzed by LPL without sample extraction. The derivatized fatty acids could be adequately separated from interfering peaks. The substrate for LPL was loaded in with a saturated concentration of 10 mM, and bovine serum albumin (BSA) was found to be a key factor in LPL reactions. Gum Arabic (GA) was chosen for the emulsifier, but the used concentration as 1% was critical. Optimum condition for measuring the LPL activity was found when the produced OA dissolved in acetonitrile (MeCN). The accuracy values for the determination of LPL activity in 10 μL of rat post heparin plasma were 108.73 - 114.36%, and the intra- and inter-day precision values were within 1.28% and 2.91%, respectively. The limit of detection was about 4.53 nM. The proposed HPLC method was successfully applied to determine LPL activity in the post heparin plasma samples of normal and streptozotocin-induced diabetic rats. The LPL activity of diabetic rat was reduced about 52.3% compared with control. The established assay system is envisioned to be useful for determining LPL activity in different physiological and pathological conditions to clarify the relationship between LPL activity and diabetes mellitus.